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Azospirillum brasilense viable cells enumeration using propidium monoazide-quantitative PCR

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Abstract

Azospirillum brasilense is a plant growth promoting bacteria used as an inoculant in diverse crops. Accurate analytical methods are required to enumerate viable cells in inoculant formulations or in planta. We developed a quantitative polymerase chain reaction (qPCR) assay associated to propidium monoazide (PMA) to evaluate the cell viability of A. brasilense in inoculant and in maize roots. A. brasilense was grown in culture medium and was exposed to 50 ℃. Maize roots were grown in vitro and harvested 7 days after inoculation. Quantification was performed by qPCR, PMA-qPCR, and plate counting. Standard curves efficiency values ranged from 85 to 99%. The limit of detection was 104 CFU per gram of fresh root. Enumeration obtained in maize roots by qPCR where higher than enumeration by PMA-qPCR and by plate counting. PMA-qPCR assay was efficient in quantifying inoculant viable cells and provides reliable results in a quickly and accurately way compared to culture-dependent methods.

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Acknowledgements

We would like to express our gratitude to Emanuel Maltempi de Souza and Roseli Prado, Universidade Federal do Paraná (UFPR) for providing A. brasilense strain FP2 and H. seropedicae strain SmR1.

Funding

This study was funded by National Council of Scientific and Technological Development (CNPq), Ministry of Science and Technology, Brazil. Elisandra T. Cunha, Ana M. Pedrolo and Mirella C. Scariot are recipients of PhD fellowships Coordination of Personnel Improvement of Higher Education (CAPES), Ministry of Education, Brazil. Franciele Paludo is recipient of Master fellowship from CNPq and Ana Carolina M. Arisi is recipient of research fellowship (PQ2) from CNPq.

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Authors and Affiliations

  1. Laboratory of Molecular Biology, Food Science and Technology Department, Federal University of Santa Catarina, Florianópolis, SC, Brazil

    Elisandra Triches da Cunha, Ana Marina Pedrolo, Franciele Paludo, Mirella Crhistine Scariot & Ana Carolina Maisonnave Arisi

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  1. Elisandra Triches da Cunha
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  2. Ana Marina Pedrolo
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  3. Franciele Paludo
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  4. Mirella Crhistine Scariot
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  5. Ana Carolina Maisonnave Arisi
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Correspondence to Ana Carolina Maisonnave Arisi.

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Communicated by Erko Stackebrandt.

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da Cunha, E.T., Pedrolo, A.M., Paludo, F. et al. Azospirillum brasilense viable cells enumeration using propidium monoazide-quantitative PCR. Arch Microbiol 202, 1653–1662 (2020). https://doi.org/10.1007/s00203-020-01877-0

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